| Literature DB >> 7929198 |
A J Baron1, C Stevens, C Wilmot, K D Seneviratne, V Blakeley, D M Dooley, S E Phillips, P F Knowles, M J McPherson.
Abstract
Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.Entities:
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Year: 1994 PMID： 7929198
Source DB: PubMed Journal: J Biol Chem ISSN： 0021-9258 Impact factor: 5.157